REDOR NMR for drug discovery

Abstract

Rotational-echo double-resonance (REDOR) NMR is a powerful and versatile solid-state NMR measurement that has been recruited to elucidate drug modes of action and to drive the design of new therapeutics. REDOR has been implemented to examine composition, structure, and dynamics in diverse macromolecular and whole-cell systems, including taxol-bound microtubules, enzyme-cofactor-inhibitor ternary complexes, and antibiotic-whole-cell complexes. The REDOR approach involves the integrated design of specific isotopic labeling strategies and the selection of appropriate REDOR experiments. By way of example, this digest illustrates the versatility of the REDOR approach, with an emphasis on the practical considerations of experimental design and data interpretation.

Keywords: Drug discovery; REDOR; Solid-state NMR; Taxol; Vancomycin; Whole-cell NMR.

Figure 1. The REDOR measurement

REDOR is performed in two parts, once with dephasing pulses (S spectrum) and once without (full echo, S₀ spectrum). REDOR spectra are typically collected with standard xy-8 phase cycling, on both observed and dephasing channels.

Figure 2. Dipolar couplings to distances

Strengths of the dipolar couplings (D) between pairs of nuclei at different distances and the percent dephasing (ΔS/S₀) that would be expected in REDOR experiments as a function of evolution time to for 2, 4, 6, and 10 Å distances between isolated spin pairs (top). Values of constants used in calculations of D (middle). Calculated plots of ΔS/S₀ for C–F, C–P, C–D, and C–N pairs at 6Å for 25 ms of dipolar evolution time (bottom).

Figure 3. A 7.8 Å ²H-¹⁹F distance in taxol

(A) ²H{¹⁹F} 64-Tr REDOR spectra of tubulin-bound taxol analogue 4, corresponding to 8 ms dipolar evolution time with MAS at 8 kHz, resulted in 8 % dephasing and a calculated distance of 7.8 (± 0.5)Å. The full-echo spectrum is shown in blue, and the dephased spectrum in red. The time domain signals were detected synchronously with the rotor so that all sidebands have been folded into the centerbands. The CD₃ peak is assigned zero frequency and the aromatic-D peak appears about 5 ppm to low field. Each spectrum resulted from the accumulation of 640,000 scans with a 1.5 sec recycle delay (approximately 11 days each and, thus, days for the ΔS spectrum). (B) ²H{¹⁹F}32-Tr REDOR spectra of tubulin-bound taxol analogue 5, corresponding to 4 ms dipolar evolution time with MAS at 8 kHz, resulted in 6 % dephasing and a calculated distance of 6.3 (± 0.5)Å. Each spectrum resulted from the accumulation of 1,056,000 scans (18 days each and, thus, 36 days for the ΔS spectrum). Spectra were obtained on a 500 MHz spectrometer. Figure adapted from Paik et al.

Figure 4. Distances between an antibiotic and the bacterial cell wall

(A) Chemical schematic of the S. aureus peptidoglycan, highlighting the unique bridge-link and crosslink sites that are often targeted in REDOR experiments through the use of selective D-[1-¹³C]Ala, [¹⁵N]Gly, and L-[ε-¹⁵N]Lys labeling. (B) Chemical structure of [¹⁹F]oritavancin. (C) ¹³C{¹⁹F} REDOR dephasing (ΔS/S₀) as a function of the dipolar evolution time, t, for complexes of [¹⁹F]oritavancin with peptidoglycan isolated from S. aureus whole cells grown on media containing D-[1-¹³C]alanine. The binding site occupancy of [¹⁹F]oritavancin complexed to cell walls was either 33% (open circles) or 16% (closed circles), wherein 2 µmol antibiotic was bound to 20 or 40 mg, respectively, of isolated peptidoglycan (dry mass). The calculated cell wall dephasing (solid lines) assumed a Gaussian distribution of distances for isolated ¹³C-¹⁹F pairs centered at 7.6 Å with a width of 1.5 Å. The dashed line also provides a reasonable fit to the data. Spectra were obtained on a 500 MHz spectrometer. Figure adapted from Kim et al.

Figure 5. Site-specific antibiotic-cell wall distances in whole cells

(A) Full echo ¹³C CPMAS and TEDOR selected spectrum of lyophilized whole cells of S. aureus grown on media containing D-[1-¹³C]Ala and [¹⁵N]Gly in the presence of an alanine racemase inhibitor to ensure selective alanine labeling. The ¹³C CPMAS spectrum (bottom) was the result of 256 scans. The TEDOR-selected ¹³C spectrum (top) was the result of 120,0scans and harbors contributions only from labeled and natural abundance carbons that are bonded to ¹⁵N. (B) ¹⁵N→ ¹³C{¹⁹F} TEDOR-selected REDOR spectra of the whole-cell sample complexed to [¹⁹F]oritavancin after 15 ms dipolar evolution time (96 rotor cycles with MAS at 6250 Hz). S₀ and S spectra resulted from the accumulation of 120,000scans. (C) ¹⁵N→¹³C{¹⁹F} TEDOR-selected REDOR dephasing (ΔS/S₀) as a function of dipolar evolution time. The calculated dephasing using a Gaussian distribution of distances centered at 7.4 Å (solid line) provided a better match to the experimental dephasing than a single 7.0 Å distance (dashed line). The dotted lines show the calculated dephasing for single distances of 6.5 Å (upper) and 7.5 Å (lower). Spectra were obtained on a 500 MHz spectrometer. (D) Molecular model of [¹⁹F]oritavancin bound to the peptidoglycan of S. aureus generated from several distance measurements including the featured TEDOR-REDOR distance. Figure adapted from Cegelski et al.

Figure 6. Cross-link density and inhibition of crosslinking in whole cells

REDOR selection and quantification of peptidoglycan crosslinks in S. aureus whole cells labeled with [¹⁵N]glycine and D-[1-¹³C]alanine and a racemase inhibitor in the absence (black) and presence of 0.15 µg/mL penicillin (blue) and 20 µg/mL oritavancin (green). The REDOR difference measures the relative number of cross-links per pentagycyl bridging segment. Penicillin has a large effect, as a known crosslinking inhibitor and oritavancin also inhibited transpeptidation. Spectra were obtained on a 300 MHz spectrometer. Figure adapted from Kim et al.

Figure 7. Lipid cartography and guest-lipid proximities

(A) ¹³C{³¹P} REDOR spectra of multilamellar vesicles containing 5% fluorinated DPPC and 95% DPPC with [3-¹³C]estradiol benzoate (left) and estradiol [1¹³C]benzoate (right) after 9 ms of of dipolar evolution (64 Tr with MAS at 7143 Hz). (B) ¹³C{¹⁹F} 128-Tr REDOR spectra of the estradiol [1-¹³C]benzoate-containing vesicles. The substantial dephasing for the label in the benzoate ring placed that carbon proximate to the lipid tails. Spectra were obtained on a 500 MHz spectrometer. Figure adapted from Cegelski et al.